This study was designed to further characterize the fibrinogen-binding properties of Actinornyces pyogenes. The fibrinogen-binding capacities of a selected A. pyogenes culture could be significantly enhanced by heat pretreatment (60 "C, 1 h) of the bacteria. The fibrinogen-binding site seemed to be a protein which was specific for fibrinogen. In phagocytosis studies, binding of fibrinogen to A. pyogenes significantly increased the phagocytic capacity of bovine polyrnorphonuclear leucocytes. ;i. Corresponding author U.S. Copyright Clearance Center Code Statement: 0931 -1793/94/4109 -0588$10.50/0 Fibrinogen-binding Properties of Actinornyces Pyogenes 589
Material and Methods
Bacterial cultureThe A . pyogenes culture used in this study (A. pyogenes 103) has been described previously (LAMMLER, 1990). This culture, originally isolated from porcine brain has been characterized biochemically and serologically (LAMMLER and BLOBEL, 1988;DING and LAMMLER, 1992). The A. pyogenes culture was grown microaerobically on Columbia blood-agar plates (Oxoid, Wesel, Germany) containing 5 % sheep blood, or in Todd Hewitt Broth (THB, Gibco, Karlsruhe, Germany) for 48 h at 37 "C in a candle jar. For comparative purposes, the fibrinogen-binding group A streptococcal culture Streptococcus pyogenes 45986 was used (CHHATWAL et al., 1985).
Binding assaysRadiolabelling: Human fibrinogen (Deutsche Kabi, Miinchen, Germany) was radiolabelled using the chloramin-T method. (HUNTER and GREENWOOD 1962). The binding assays were performed with 20 pl (40 ng; 20 000-40 000 ~pm)'~'I-fibrinogen as described recently (CHHATWAL et a]., 1985).Heat treatment: For heat treatment, the cells, suspended in 0.25 mol/1 phosphate buffered saline (PBS), p H 7.5, were exposed to 60 or 95 "C for different time intervals. Heat-treated cells were subsequently used in the binding assays.Guanidinium extraction: Following the method described by RUSSEL and FACKLAM (1975), the bacteria were cultivated, resuspended in 6 M guanidinium hydrochloride (Merck, Darmstadt, Germany) (10 % of the original cultivation volume) and incubated for 1 h at room temperature with shaking. After washing (3 X ) with PBS, the bacteria were adjusted photometrically to approximately lo9 bacteria/ml (10 % transmission at 620 nm, Bausch and Lomb, Rochester, NY, USA) and used in the binding assay (CHHATWAL et al., 1984).Ultrasonication: For ultrasonication, the bacteria were cultivated, resuspended in PBS and sonicated (20 kHz, Sonifier B12, Branson Sonic Power, Danbury, USA) five times for 10 s. After washing (twice with PBS), the bacteria were photometrically adjusted and used in the binding assay.
Pronase, and trypsin treatment:For enzyme treatment, the photometrically adjusted bacteria (lo9 bacteria/ml) were suspended in PBS, incubated with 50 pg of pronase (Merck) and trypsin (Merck), and incubated for 1 h at 37 "C. The bacteria were washed with PBS and subsequently used in the binding assay (LAMMLER et al., 1983).
